Factors regulating template switch in vitro by viral RNA-dependent RNA polymerases: Implications for RNA���RNA recombination

Copy-choice RNA recombination occurs during viral RNA synthesis when the viral transcription complex switches templates. We demonstrate that RNA-dependent RNA polymerase from bovine viral diarrhea virus and the replicases from three plant-infecting RNA viruses can produce easily detectable recombination products in vitro by switching templates during elongative RNA synthesis. Template sequence and/or structure, and NTP availability affected the frequency of template switch by the transcription complex. Our results provide biochemical support for copy-choice recombination and establish assays for mechanistic analyses of intermolecular RNA recombination in vitro.

In vitro reconstitution of human replication factor C from its five subunits.

Replication factor C (RFC, also called Activator I) is part of the processive eukaryotic DNA polymerase holoenzymes. The processive elongation of DNA chains requires that DNA polymerases are tethered to template DNA at primer ends. In eukaryotes the ring-shaped homotrimeric protein, proliferating cell nuclear antigen (PCNA), ensures tight template-polymerase interaction by encircling the DNA strand. Proliferating cell nuclear antigen is loaded onto DNA through the action of RFC in an ATP-dependent reaction. Human RFC is a protein complex consisting of five distinct subunits that migrate through SDS/polyacrylamide gels as protein bands of 140, 40, 38, 37, and 36 kDa. All five genes encoding the RFC subunits have been cloned and sequenced. A functionally identical RFC complex has been isolated from Saccharomyces cerevisiae and the deduced amino acid sequences among the corresponding human and yeast subunits are homologous. Here we report the expression of the five cloned human genes using an in vitro coupled transcription/translation system and show that the gene products form a complex resembling native RFC that is active in supporting an RFC-dependent replication reaction. Studies on the interactions between the five subunits suggest a cooperative mechanism in the assembly of the RFC complex. A three-subunit core complex, consisting of p36, p37, and p40, was identified and evidence is presented that p38 is essential for the interaction between this core complex and the large p140 subunit.

The CuA center of cytochrome-c oxidase: electronic structure and spectra of models compared to the properties of CuA domains.

The electronic structure and spectrum of several models of the binuclear metal site in soluble CuA domains of cytochrome-c oxidase have been calculated by the use of an extended version of the complete neglect of differential overlap/spectroscopic method. The experimental spectra have two strong transitions of nearly equal intensity around 500 nm and a near-IR transition close to 800 nm. The model that best reproduces these features consists of a dimer of two blue (type 1) copper centers, in which each Cu atom replaces the missing imidazole on the other Cu atom. Thus, both Cu atoms have one cysteine sulfur atom and one imidazole nitrogen atom as ligands, and there are no bridging ligands but a direct Cu-Cu bond. According to the calculations, the two strong bands in the visible region originate from exciton coupling of the dipoles of the two copper monomers, and the near-IR band is a charge-transfer transition between the two Cu atoms. The known amino acid sequence has been used to construct a molecular model of the CuA site by the use of a template and energy minimization. In this model, the two ligand cysteine residues are in one turn of an alpha-helix, whereas one ligand histidine is in a loop following this helix and the other one is in a beta-strand.

NGC 7419 as a template for red supergiant clusters

Context. The open cluster NGC 7419 is known to contain five red supergiants and a very high number of Be stars. However, there are conflicting reports about its age and distance that prevent a useful comparison with other clusters. Aims. We intend to obtain more accurate parameters for NGC 7419, using techniques different from those of previous authors, so that it may be used as a calibrator for more obscured clusters. Methods. We obtained Str��mgren photometry of the open cluster NGC 7419, as well as classification spectroscopy of ~20 stars in the area. We then applied standard analysis and classification techniques. Results. We find a distance of 4 �� 0.4 kpc and an age of 14 �� 2 Myr for NGC 7419. The main-sequence turn-off is found at spectral type B1, in excellent agreement. We identify 179 B-type members, implying that there are more than 1200 M��� in B stars at present. Extrapolating this to lower masses indicates an initial cluster mass of between 7000 and 10���000 M���, depending on the shape of the initial mass function. We find a very high fraction (���40%) of Be stars around the turn-off, but very few Be stars at lower masses. We also report for the first time a strong variability in the emission characteristics of Be stars. We verified that the parameters of the red supergiant members can be used to obtain accurate cluster parameters. Conclusions. NGC 7419 is sufficiently massive to serve as a testbed for theoretical predictions and as a template to compare more obscured clusters. The distribution of stars above the main-sequence turn-off is difficult to accommodate with current evolutionary tracks. Though the presence of five red supergiants is marginally consistent with theoretical expectations, the high number of Be stars and very low number of luminous evolved B stars hint at some unknown physical factor that is not considered in current synthesis models.

Cycloadditions of isobenzofuran to a constrained template bearing neighboring dienophiles

A high yielding synthesis of the pentacyclic diene-dione 1 has enabled investigation of its reactivity as a double dienophile in Diels-Alder [4+2] cycloadditions with isobenzofuran, leading to novel and highly symmetrical three-sided cavitands 3 and 4.

Magnetosomal matrix: ultrafine structure may template biomineralization of magnetosomes

The organic matrix surrounding bullet-shaped, cubo-octahedral, D-shaped, irregular arrowhead-shaped, and truncated hexa-octahedral magnetosomes was analysed in a variety of uncultured magnetotactic bacteria. The matrix was examined using low- (80 kV) and intermediate- (400 kV) voltage TEM. It encapsulated magnetosomes in dehydrated cells, ultraviolet-B-irradiated dehydrated cells and stained resin-embedded fixed cells, so the apparent structure of the matrix does not appear to be an artefact of specimen preparation. High-resolution images revealed lattice fringes in the matrix surrounding magnetite and greigite magnetosomes that were aligned with lattice fringes in the encapsulated magnetosomes. In all except one case, the lattice fringes had widths equal to or twice the width of the corresponding lattice fringes in the magnetosomes. The lattice fringes in the matrix were aligned with the {311}, {220}, {331}, {111} and {391} related lattice planes of magnetite and the {222} lattice plane of greigite. An unidentified material, possibly an iron hydroxide, was detected in two immature magnetosomes containing magnetite. The unidentified phase had a structure similar to that of the matrix as it contained {311}, {220} and {111} lattice fringes, which indicates that the matrix acts as a template for the spatially controlled biomineralization of the unidentified phase, which itself transforms into magnetite. The unidentified phase was thus called pre-magnetite. The presence of the magnetosomal matrix explains all of the five properties of the biosignature of the magnetosomal chain proposed previously by Friedmann et al. and supports their claim that some of the magnetite particles in the carbonate globules in the Martian meteorite ALH84001 are biogenic. Two new morphologies of magnetite magnetosomes are also reported here (i.e. tooth-shaped and hexa-octahedral magnetosomes). Tooth-shaped magnetite magnetosomes elongated in the [110] direction are reported, and are distinct from arrowhead-shaped and bullet-shaped magnetosomes. Elongation of magnetite magnetosomes in the [110] direction has not been reported previously. A Martian hexa-octahedral magnetite particle was previously characterized by Thomas-Keptra et al. and compared with truncated hexa-octahedral magnetite magnetosomes. Hexa-octahedral magnetite magnetosomes with the same morphology and similar sizes and axial ratios as those reported by Thomas-Keptra et al. are characterized here. These observations support their claim that ALH84001 contains evidence for a past Martian biota.

The Mental Health Policy Template: Domains and elements for mental health policy formulation

Mental disorders are a major and rising cause of disease burden in all countries. Even when resources are available, many countries do not have the policy and planning frameworks in place to identify and deliver effective interventions. The World Health Organization (WHO) and the World Bank have emphasized the need for ready access to the basic tools for mental health policy formulation, implementation and sustained development. The Analytical Studies on Mental Health Policy and Service Project, undertaken in 1999-2001 by the International Consortium for Mental Health Services and funded by the Global Forum for Health Research aims to address this need through the development of a template for mental health policy formulation. A mental health policy template has been developed based on an inventory of the key elements of a successful mental health policy. These elements have been validated against a review of international literature, a study of existing mental health policies and the results of extensive consultations with experts in the six WHO regions of the world. The Mental Health Policy Template has been revised and its applicability will be tested in a number of developing countries during 2001-2002. The Mental Health Policy Template and the work of the Consortium for Mental Health Services will be presented and the future role of the template in mental health policy development and reform in developing countries will be discussed.

The cyclotide family of circular miniproteins: Nature's combinatorial peptide template

The cyclotides are a recently discovered family of miniproteins that contain a head-to-tail cyclized backbone and a knotted arrangement of disulfide bonds. They are approximately 30 amino acids in size and are present in high abundance in plants from the Violaceae, Rubiaceae, and Cucurbitaceae families, with individual plants containing a suite of up to 100 cyclotides. They have a diverse range of biological activities, including uterotonic, anti-HIV, antitumor, and antimicrobial activities, although their natural function is likely that of defending their host plants from pathogens and pests. This review focuses on the structural aspects of cyclotides, which may be thought of as a natural combinatorial peptide template in which a wide range of amino acids is displayed on a compact molecular core made up of the cyclic cystine knot structural motif. Cyclotides are exceptionally stable and are resistant to denaturation via thermal, chemical, or enzymatic treatments. The struclural features that contribute to their remarkable stability are described ill this review. (c) 2006 Wiley Periodicals, Inc.

Uma Introdu����o �� Sintaxe do C++: C++03 e C++11

A interac����o dos humanos com os computadores envolve uma combina����o das tarefas de programa����o e de utiliza����o. Nem sempre �� expl��cita a diferen��a entre as duas tarefas. Introduzir comandos num programa de desenho assistido por computador �� utiliza����o ou programa����o numa linguagem interpretada? Modificar uma folha de c��lculo com macros �� utiliza����o ou programa����o? Usar um ���Integrated Development Environment��� ou IDE para inserir dados num ficheiro �� utiliza����o (do IDE) ou programa����o? A escrita de um texto usando LaTeX ou HTML �� utiliza����o ou programa����o numa ���markup language���? Recorrer a um programa de computa����o simb��lica �� utiliza����o ou programa����o? Utilizar um processador de texto �� utiliza����o ou programa����o visual? Ao utilizador n��o se exige um conhecimento completo de todos os comandos, todos os menus, todos os s��mbolos do software que utiliza. Nem a memoriza����o da sintaxe e de todos os pormenores de funcionamento de um programa �� um atributo necess��rio ou sequer ��til ao utilizador; a concretiza����o desse conhecimento n��o assegura maior efici��ncia na utiliza����o. Quando se come��a, apenas algumas instru����es elementares s��o recebidas, por vezes de um colega, de um Professor, ou obtidas recorrendo �� pesquisa na Internet. Com a familiariza����o, o utilizador exige mais do Software que usa e de si pr��prio: um manual passa a ser um recurso de grande utilidade. A confian��a conquistada gera, periodicamente, a necessidade de auto-exame e de aumento do ��mbito do conhecimento. Desta forma, quem utiliza computadores acaba por ser confrontado com uma tarefa que, efectivamente, pode ser considerada ou requer programa����o. P��e-se uma quest��o no imediato (se ningu��m decidiu por si) que �� a da selec����o da linguagem de programa����o. A abordagem multiparadigma e longa experi��ncia de utiliza����o do C++ tornam-no atractivo para aplica����es onde a efici��ncia se combina com a disponibilidade de estruturas de dados e algoritmos adoptados pela ind��stria (o que coloquialmente se denomina STL, Standard Template Library, cf. [#breymann, #josuttis], mais geralmente biblioteca Standard). Adicionalmente, linguagens populares como o Java, C# e PHP possuem sintaxes inspiradas e em muitas partes coincidentes com as do C e C++. Por exemplo, um ciclo ���for��� em Java �� parcialmente coincidente com o do C99, que �� um sub-conjunto do ���for��� do C++. S��o os pormenores, a efici��ncia e as capacidades do C++ que permitem a cria����o de software Profissional. Todos os sistemas operativos cl��ssicos (Unix, Microsoft Windows, Linux) disp��em de compiladores, IDE, bibliotecas e s��o em grande parte constru��dos recorrendo a C e C++. Relativamente a outras linguagens, a quantidade de ferramentas dispon��vel e o conhecimento adquirido durante d��cadas �� dif��cil de ignorar. Esse conhecimento faz com que a sintaxe do C++ pare��a muito maior do que o estritamente necess��rio e afaste potenciais interessados. A longa evolu����o do C++ introduziu tamb��m uma diferen��a no estilo muito marcada. C��digo dos anos 80 e 90 do s��culo XX �� frequentemente menos leg��vel do que o que correntemente se produz. Muitos tutoriais dispon��veis online fazem parecer a linguagem menos rigorosa (e mais complexa) do que na realidade ��, j�� que raramente �� apresentado o caso geral da sintaxe. Constata-se que muitos autores ainda usam os cabe��alhos do C, quando j�� n��o s��o necess��rios. Scott Meyers afirma que o C++ �� uma federa����o de linguagens [#scottmeyers] e por esse facto requer perspectivas de abordagem distintas de outras linguagens. Sem alguma sistematiza����o �� dif��cil apreciar a sua compacidade e coer��ncia. Por��m, a forma harmoniosa como as componentes sint��cticas se encaixam �� uma grande mais-valia do C++ s�� constatada com experimenta����o e leitura atenta. A presente monografia dirige-se a quem pretenda utilizar o C++ como ferramenta profissional de Software. Em termos de pr��-requisitos Acad��micos, dir-se-�� que um curso (1�� Ciclo) de Ci��ncia ou de Engenharia aumentar�� o interesse por certos aspectos mais t��cnicos da linguagem mas qualquer indiv��duo com gosto pela experimenta����o tirar�� proveito do conte��do. Este texto n��o busca a exaustividade enciclop��dica na cobertura do tema. Neste texto forne��o, de forma directa, uma introdu����o ao C++ a qual permite come��ar a produzir c��digo sem os custos da dispers��o de fontes e nota����es na recolha de informa����o. Antecipo assim a sua utiliza����o nos Pa��ses de L��ngua Portuguesa, uma vez que os textos que encontrei s��o ora mais exigentes ora menos completos, frequentemente ambos.

Open Specification for BIM : Sydney Opera House Case Study

Executive Summary The objective of this report was to use the Sydney Opera House as a case study of the application of Building Information Modelling (BIM). The Sydney opera House is a complex, large building with very irregular building configuration, that makes it a challenging test. A number of key concerns are evident at SOH: ��� the building structure is complex, and building service systems - already the major cost of ongoing maintenance - are undergoing technology change, with new computer based services becoming increasingly important. ��� the current ���documentation��� of the facility is comprised of several independent systems, some overlapping and is inadequate to service current and future services required ��� the building has reached a milestone age in terms of the condition and maintainability of key public areas and service systems, functionality of spaces and longer term strategic management. ��� many business functions such as space or event management require up-to-date information of the facility that are currently inadequately delivered, expensive and time consuming to update and deliver to customers. ��� major building upgrades are being planned that will put considerable strain on existing Facilities Portfolio services, and their capacity to manage them effectively While some of these concerns are unique to the House, many will be common to larger commercial and institutional portfolios. The work described here supported a complementary task which sought to identify if a building information model ��� an integrated building database ��� could be created, that would support asset & facility management functions (see Sydney Opera House ��� FM Exemplar Project, Report Number: 2005-001-C-4 Building Information Modelling for FM at Sydney Opera House), a business strategy that has been well demonstrated. The development of the BIMSS - Open Specification for BIM has been surprisingly straightforward. The lack of technical difficulties in converting the House���s existing conventions and standards to the new model based environment can be related to three key factors: ��� SOH Facilities Portfolio ��� the internal group responsible for asset and facility management - have already well established building and documentation policies in place. The setting and adherence to well thought out operational standards has been based on the need to create an environment that is understood by all users and that addresses the major business needs of the House. ��� The second factor is the nature of the IFC Model Specification used to define the BIM protocol. The IFC standard is based on building practice and nomenclature, widely used in the construction industries across the globe. For example the nomenclature of building parts ��� eg ifcWall, corresponds to our normal terminology, but extends the traditional drawing environment currently used for design and documentation. This demonstrates that the international IFC model accurately represents local practice for building data representation and management. ��� a BIM environment sets up opportunities for innovative processes that can exploit the rich data in the model and improve services and functions for the House: for example several high-level processes have been identified that could benefit from standardized Building Information Models such as maintenance processes using engineering data, business processes using scheduling, venue access, security data and benchmarking processes using building performance data. The new technology matches business needs for current and new services. The adoption of IFC compliant applications opens the way forward for shared building model collaboration and new processes, a significant new focus of the BIM standards. In summary, SOH current building standards have been successfully drafted for a BIM environment and are confidently expected to be fully developed when BIM is adopted operationally by SOH. These BIM standards and their application to the Opera House are intended as a template for other organisations to adopt for the own procurement and facility management activities. Appendices provide an overview of the IFC Integrated Object Model and an understanding IFC Model Data.